| Tobacco-specific nitrosamines(TSNAs)is one harmful carcinogen and nitrate is one important precursors of TSNA formation.The concentration of TSNAs in burley tobacco is higher than that in flue-cured tobacco,which is negative to improve security of tobacco.In this present study,field experiments and pot experiments were carried out to investigate differences of nitrate accumulation and metabolism and NO3-uptake dynamics in burley and flue-cured tobacco to supply theories of reducing nitrate and TSNAs in burley tobacco leaves.And metabolism,reasons and mechanisms of nitrate accumulation in tobacco leaves were explored,and one effective methods of regulating nitrate and TSNAs accumulation were screened out.The main conclusions are as following:1)Differences of root morphology and NO3-uptake dynamics between burley tobacco and flue-cured tobacco were studied and the relationship between nitrate accumulation and parameters of root nitrate uptake was analyzed at the same nitrogen application level.The results were showed that root length,root surface,root volume and root tips were higher in burley tobacco than in flue-cured tobacco,and maximum value of NO3-uptake rate and amount was lower in burley tobacco than in flue-cured tobacco.Being relative to flue-cured tobacco,pigment content,photosynthesis,dry matter accumulation,carbohydrate content and nitrogen accumulation in whole plant,nitrate content,total nitrogen content and ratio of nitrate nitrogen to total nitrogen content were significantly higher in burley tobacco.The results demonstrated that the development of root morphology in burley tobacco was better than that in flue-cured tobacco,but the ability of absorbing in burley tobacco was not great,which was indicated that the nitrate accumulation might be little correlated with the ability of absorbing in root.2)To make clear that the pattern and key period of nitrate accumulation in burley tobacco and its effects to the formation of TSNAs,the change patterns of nitrogen metabolism and nitrate content and relationships between nitrate,nitrite,alkaloid content and TSNA formation in burley tobacco and flue-cured tobacco were studied at the same yield conditions.The results showed that pigment content,ratio of nitrate reductase activity(NRA)to nitrogen application,glutamine synthetase activity(GSA)and soluble protein content in burley tobacco were significantly lower than that in flue-cured tobacco,and nitrate,total nitrogen and TSNAs concentration were significantly higher in burley tobacco than in flue-cured tobacco.Correlation analysis was showed that the relationship between TSNAs content andnitrate content was significant,indicating that high nitrate accumulation was the important factor of high TSNAs formation.During the development of tobacco,change pattern of nitrate content were increasing firstly and then decreasing,and the maximum value of change pattern was determined in the middle of fast-growth period and parameter were decreasing slowly at maturity stage.The fast-growth period and the maturity stage were important to decreasing nitrate accumulation and differences between burley tobacco and flue-cured tobacco in nitrate content were happened in seedlings.Nitrate content was correlation with GSA in tobacco leaves in fast-growing period and at maturity stage,indicating that nitrate accumulation in burley tobacco leaves was correlated with ability of nitrogen reducing and assimilation during tobacco leaf development process.To sum up,weak ability of nitrogen reducing and assimilation was the significant reasons for nitrate accumulation,and high nitrate accumulation lead to high TSNA formation,indicating that enhancing nitrogen assimilation is the key method to decreas nitrate accumulation.3)To explore the reasons and mechanisms of nitrate accumulation in burley tobacco,differences of carbon and nitrogen metabolism pathway and its product in burley tobacco and flue-cured tobacco were analyzed and relationships between nitrate and some key parameters were studied by technology of biochemical,physiological and transcriptomic at the same nitrogen application and the same leaf biomass accumulation conditions.The results showed that different expression genes(DEGs)between burley tobacco and flue-cured tobacco in RNA-Seq were screened out by STEM,and then analysis of GO and KEGG were carried out to pinpoint the direction leading to nitrate accumulation in burley tobacco.The cellular-component of DEGs was focus on photosystem ? and photosystem ?,and the DEGs correlated with sucrose and starch biosynthesis(AGPS1?SPS2?SUS2?DPE2?TPS11),NLP7 and nitrogen transport and assimilation(NPF3.1?NPF7.3?NRT2.1?NRT2.5?NIA1?NIA2?GS1?GDHA)were constantly down-regulated,which was consist with the change of dry matter accumulation and carbon nitride compounds,indicating that these several DEGs were the key genes causing nitrate accumulation in burley tobacco.The results determined on the basic of biochemical and physiological were showed that pigment and reducing soluble sugar content and photosynthetic rate were lower of 41.72 %,42.89 % and 33.44 % in burley tobacco than in flue-cured tobacco,respectively,and total nitrogen and nitrate content were higher of 32.51 % and 103.36 % in burley tobacco than in in flue-cured tobacco at the same nitrogen level,respectively.Correlation between NO3-N content and NRA,GSA,pigment content and photosynthetic rate was significantly positive,indicating that nitrate content in tobacco leaves was correlated with activities of nitrogen assimilation and photosynthesis.With spraying sodium molybdate,nitrogen assimilation increased significantly and nitrate content decreased significantly.After spraying glycerol,carbohydrats content increased and nitrate content decreased significantly with nitrogen assimilationenhanced.Weak ability of nitrogen assimilation might be the reason of nitrate accumulation,and reduced ability of carbon fixation and sugars biosynthesis caused the insufficient of energy and reducing power,which might lead to nitrate accumulation in burley tobacco leaves.4)In our previous work,effects of spraying different modulators on regulation of nitrate metabolism and TSNAs accumulation were studied to screen out one effective method.The results showed that NRA and soluble protein content in upper leaves increased by 19.30 % and 5.92 % with spraying sodium molybdate 14 days later in fast growth period,and GSA and soluble protein content in upper leaves decreased by 18.79 % and 24.54 % by spraying glufosinate 14 days later at maturity stage.After spraying sodium molybdate during fast growth period and the sodium molybdate and glufosinate combination at maturity stage,GSA,ammonia volatilization rate,nitrate and TSNAs concentration in tobacco leaves had significantly decreased by 84.48 %,49.65 %,47.00 % and 51.76 %,respectively,indicating that ability of nitrate reducing had enhanced by spraying sodium molybdate in fast growth period,meanwhile,the loss of ammonia volatilization was promoted to realize the decrease of nitrate accumulation at maturity stage,which was one effective method of reducing nitrate and TSNAs content in burley tobacco.After spraying glycerol(concentration,from 0.025 % to 0.15 %),nitrate had significantly decreased in burley tobacco seedling leaves,and effects of 0.1 % glycerol were best.With spraying glycerol,GSA,pigment and soluble protein content,leaf biomass and biomass aboveground part,and total soluble sugar and reducing soluble sugar content in burley tobacco leaves had increased significantly and total nitrogen and nitrate content decreased significantly.Analysis of correlation showed that reducing folds of nitrate were significantly correlated with the increasing folds of pigment and GSA in burley tobacco,indicating that glycerol had promoted the ability of nitrogen assimilation and reduced nitrate accumulation.Insufficient of carbon source in burley tobacco might be one effective modulator of reducing nitrate accumulation. |
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